Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression

Abstract

The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active / inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies. We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method, is opening new avenues to understand how self-assembly of phase separated nuclear bodies possibly contributes to mammalian genome organization and gene expression.

Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

“…To investigate the role played by LLPS in spatial genome organization, we used human HeLa cells that were treated with 1,6-HD. This aliphatic alcohol is predicted to disrupt weak hydrophobic interactions, both in vitro and in vivo , disassembling LLPS-dependent macromolecular condensates ( 61–63 ). Molecular condensates depended purely on phase separation driven by electrostatic interactions are expected to be unaffected by 1,6-HD treatment ( 61 ).…”

“…This aliphatic alcohol is predicted to disrupt weak hydrophobic interactions, both in vitro and in vivo , disassembling LLPS-dependent macromolecular condensates ( 61–63 ). Molecular condensates depended purely on phase separation driven by electrostatic interactions are expected to be unaffected by 1,6-HD treatment ( 61 ). Nevertheless, 1,6-HD is the only available tool to test in vivo the contribution of LLPS in the assembly of macromolecular complexes at this moment ( 63 ).…”

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during development

“…Recent studies reported the existence of enhancer hubs: spatially-localized clusters containing multiple enhancers 27,28,31 that may facilitate transcriptional activation by creating a local microenvironment whereby transcriptional resources are shared, akin to early models of 'transcription hubs' 79 . Formation of enhancer hubs may require interactions between components of the transcriptional machinery which could contribute, or result from, the assembly of phase-separated condensates 30,35,36,[80][81][82] . In this model, enhancers need not directly touch their target promoters but merely come into close proximity 24,83 .…”

Drosophila insulator proteins exhibit in-vivo liquid-liquid phase separation properties

“…Therefore, to obtain further insights into the nature of the chemical interactions underlying assembly of insulator proteins, we looked at the effect of 1,6-hexanediol (1,6-HD) on insulator bodies. 1,6-HD is an agent that perturbs hydrophobicity-dependent LLPS condensates presumably through disruption of weak hydrophobic interactions (41, 74, 75). In addition, unlike LLPS entities like the nucleolus (76) and transcription condensates (77), solid aggregates such as viral replication compartments (78), the cytoskeleton(74) and tetO binding (79) are largely resistant to 1,6-HD.…”